Our laboratory has developed a technique for selecting for transformed bone marrow cells in intact mice by utilizing drug resistance genes and drug treatment. Apart from being a unique means of inserting new genetic information into living animals, this technique permits investigation of methods for developing enhanced effectiveness of anti-cancer drugs and gene replacement therapy of such genetic diseases as sickle cell disease and thalassemia. We are proposing to investigate several methods of transformation in order to develop optimal techniques for use in intact animals. These methods involve calcium precipitated DNA, cell-cell fusion with polyethylene glycol and Sendai virus, and the use of micro-cell techniques. Purified herpes virus thymidine kinase gene in a variety of forms (E. coli derived plasmid pBR322, free gene, ligated gene) will be used to confer resistance to the folate antagonist methotrexate in cell lines and mouse hematopoietic cells, and also as a means of introducing other genetic information, particularly the human Beta-globin gene into host cellular DNA. The above methods will be investigated comparing mouse thymidine kinase negative fibroblasts (LTK-cells) and wild type cells. Factors influencing the transformation process such as the necessity for continued drug selection and the stability of foreign DNA introduced into host cells will be investigated. An analysis will be made of cell lines and spleens from transformed animals using hybridization kinetics of complementary strands of DNA to determine the presence of viral gene sequences. Finally, further studies will attempt transformation using tissues other than bone marrow from intact animals.